Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the β-subunit flap domain of Escherichia coli RNA polymerase The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Twist, K.-A. F., E. A. Campbell, P. Deighan, S. Nechaev, V. Jain, E. P. Geiduschek, A. Hochschild, and S. A. Darst. 2011. “Crystal Structure of the Bacteriophage T4 Late-Transcription Coactivator gp33 with the -Subunit Flap Domain of Escherichia Coli RNA Polymerase.” Proceedings of the National Academy of Sciences 108 (50): 19961– 66. https://doi.org/10.1073/pnas.1113328108. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41483152 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Crystal structure of the bacteriophage T4 late- transcription coactivator gp33 with the β-subunit flap domain of Escherichia coli RNA polymerase Kelly-Anne F. Twista,1, Elizabeth A. Campbella, Padraig Deighanb, Sergei Nechaevc,2, Vikas Jainc,3, E. Peter Geiduschekc, Ann Hochschildb, and Seth A. Darsta,4 aLaboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065; bDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; and cDivision of Biological Sciences, Section of Molecular Biology, University of California, San Diego, La Jolla, CA 92093 Edited by Sankar Adhya, National Institutes of Health, NCI, Bethesda, MD, and approved September 27, 2011 (received for review August 13, 2011) Activated transcription of the bacteriophage T4 late genes, which holoenzyme accurately initiates low level (basal) transcription is coupled to concurrent DNA replication, is accomplished by an in- from T4 late promoters (13), which comprise only a −10-like mo- itiation complex containing the host RNA polymerase associated tif with consensus TATAAATA (14). Gp55 lacks a σ4-equivalent with two phage-encoded proteins, gp55 (the basal promoter domain. Instead, gp33 (112 amino acids) binds to the β-flap (11), 70 specificity factor) and gp33 (the coactivator), as well as the DNA- in competition with σ 4 (15). Gp33 has no recognizable homol- mounted sliding-clamp processivity factor of the phage T4 repli- ogy with other proteins (including σ4) and does not bind DNA; some (gp45, the activator). We have determined the 3.0 Å-resolu- instead it represses gp55-dependent basal transcription (5, 16). tion X-ray crystal structure of gp33 complexed with its RNA However, in the presence of DNA-mounted sliding-clamp gp45, polymerase binding determinant, the β-flap domain. Like domain gp55-dependent transcription is activated more than 1,000-fold σ σ β 4 of the promoter specificity factor ( 4), gp33 interacts with RNA through a direct protein/protein interaction with the -flap-bound polymerase primarily by clamping onto the helix at the tip of the gp33 (17). This requirement of the sliding-clamp for activation β σ -flap domain. Nevertheless, gp33 and 4 are not structurally couples T4 late transcription to DNA replication (18). related. The gp33/β-flap structure, combined with biochemical, Other requirements for activated late transcription include biophysical, and structural information, allows us to generate a the phage-encoded sliding-clamp loader (gp44/62), ATP or dATP structural model of the T4 late promoter initiation complex. The hydrolysis, and a DNA template with a properly placed single- model predicts protein/protein interactions within the complex strand break or end, all needed to load the gp45 sliding-clamp that explain the presence of conserved patches of surface-exposed onto DNA. The single-strand break, which serves as the sliding- residues on gp33, and provides a structural framework for inter- clamp loading site, must be on the nontranscribed strand (so that preting and designing future experiments to functionally charac- the sliding-clamp is loaded in the proper orientation), and has the terize the complex. properties of an enhancer, in that it can be placed kilobases away BIOCHEMISTRY from the promoter. The enhancer acts strictly in cis and requires ∣ Gp33 ∣ replication-coupled gene expression ∣ X-ray crystallography a continuous, unobstructed DNA path between itself and the promoter (reviewed in ref. 6). ranscription initiation in bacteria depends on the RNA poly- In this scheme, gp55 acts as the basal promoter specificity α ββ0ω factor, gp45 acts as a DNA-tracking transcriptional activator, Tmerase (RNAP) catalytic core (subunit composition 2 ) and the promoter specificity subunit σ, which combine to create and the single-strand break acts as an unusual, enhancer-like op- the RNAP holoenzyme. The σ subunit recruits RNAP to promo- erator. The properties of gp33 fit perfectly to the definition of a ters through sequence-specific interactions with two conserved coactivator (19). hexameric DNA sequence motifs, the −10 and −35 promoter Here, we present the X-ray crystal structure of gp33 bound Eco β ’ elements. The −10 element is recognized by structural domain to its binding determinant on RNAP, the -subunit s flap 2ofσ (σ2), positioned on the clamp helices of the RNAP β′-sub- domain, allowing us to compare gp33 structural and functional σ unit, while the −35 element is recognized by σ4 positioned on the characteristics to those of 4 and revealing the basis for many flap-tip-helix (FTH) of the RNAP β-subunit flap domain (1, 2). functional characteristics of gp33, including its repression of basal Transcription from weak promoters may be modulated by activa- tors that typically bind to specific DNA sequences, or operators, Author contributions: K.-A.F.T., P.D., A.H., and S.A.D. designed research; K.-A.F.T., E.A.C., located upstream of the −10∕−35 promoter elements, and sta- P.D., S.N., V.J., and S.A.D. performed research; E.P.G. contributed new reagents/analytic bilize the initiation complex through protein/protein interactions tools; K.-A.F.T., E.A.C., P.D., E.P.G., A.H., and S.A.D. analyzed data; and K.-A.F.T., E.A.C., directly with the RNAP holoenzyme, often with the α-subunit A.H., and S.A.D. wrote the paper. C-terminal domain or the σ4∕β-flap subcomplex (3). The authors declare no conflict of interest. Activated transcription of the bacteriophage T4 late genes, This article is a PNAS Direct Submission. which couples transcription of more than one-third of the phage Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, genome to concurrent DNA replication, is accomplished by a www.pdb.org (PDB ID code 3TBI). mechanism that is apparently unique (4). Activation requires the 1Present address: The Protein Crystallography Unit, ARC Center of Excellence in Structural function of two phage-encoded RNAP-binding proteins, gp55 and Functional Microbial Genomics, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia. and gp33, as well as DNA-mounted gp45, the sliding-clamp pro- 2Present address: Laboratory of Molecular Carcinogenesis, NIEHS, NIH, Research Triangle cessivity factor for the phage T4 replisome (5, 6). Both gp33 and Park, NC 27709. gp55 interact with the sliding-clamp via a hydrophobic and acidic 3Present address: Department of Biology, Indian Institute of Science Education and C-terminal tail, the sliding-clamp binding motif (SCBM) that is Research Bhopal, ITI (Gas Rahat) bulding, Govindpura, Bhopal 462023, Madhya Pradesh, attached to the body of each protein through a linker (7, 8). India. 70 Gp55 is a highly diverged member of the σ family (9, 10) that 4To whom correspondence should be addressed. E-mail: [email protected]. Escherichia coli Eco β′ binds to ( ) RNAP core on the -subunit This article contains supporting information online at www.pnas.org/lookup/suppl/ 70 clamp helices (11, 12), in competition with σ 2. The Gp55- doi:10.1073/pnas.1113328108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1113328108 PNAS ∣ December 13, 2011 ∣ vol. 108 ∣ no. 50 ∣ 19961–19966 transcription in the absence of gp45, as well as details of its inter- (residues 891–912) is composed of the FTH (residues 897–906) action with the β-flap. Finally, the new structure, in combination that is connected to the FW by two flexible loops. The FW (re- with other information, allows us to generate a model of the T4 sidues 831–890∕913–937∕1;043–1;057) aligns well with the corre- late promoter activated transcription complex that provides a sponding residues in previously solved structures of bacterial structural framework for interpreting previous results, and serves RNAP, from Thermus aquaticus (24) and Thermus thermophilus as a guide for the design of further experiments. (25), with rmsd over α-carbon positions of 1 Å and 0.9 Å, respec- tively. The FT forms a flexible appendage, which is conserved in Results and Discussion fold but found in different orientations in various structures (26). Crystallization and Structure Determination of the gp33/β-flap Com- The structure of βi9 (22), a eubacterial lineage-specific insert of plex. Gp33 binds the FTH of the Eco RNAP β-flap domain, and unknown function, has been described previously (23). The βi9 this interaction is required both for repression of gp55-mediated does not interact with gp33 (Fig. 1), nor is it involved in gp33 basal transcription by gp33 and gp45-mediated transcriptional ac- function (Fig. S2), so it is not discussed further here. tivation (15). To elucidate the details of the gp33/RNAP interac- The entire β-flap was built into the electron density map, but tion, we determined the crystal structure of the gp33/β-flap there was no clear density for two terminal segments of gp33, – complex.